铁皮石斛2个F家族ABC转运蛋白基因的克隆和表达研究

发布时间：2018-06-29 03:12

本文选题：铁皮石斛 + ATP-binding　；参考：《中草药》2017年15期

【摘要】：目的克隆铁皮石斛Dendrobium officinale ABC(ATP-binding cassette)转运蛋白F家族基因并进行生物信息学分析。方法利用RACE从铁皮石斛叶片cDNA中分离ABC基因,并进行编码蛋白相对分子质量、等电点、结构域、信号肽、跨膜域及亚细胞定位等生物信息学分析;采用DNASTAR和MEGA6进行氨基酸多序列比对和分子进化分析;借助实时荧光定量PCR技术检测基因组织表达模式。结果从铁皮石斛中分离到2个F家族ABC转运蛋白基因DoABCF1和DoABCF2(Gen Bank注册号KU160474和KU160475),全长为2 104 bp和2 193 bp,各编码1条由600和659个氨基酸组成的肽链,相对分子质量67 030和74 140,等电点6.20和5.71;DoABCF1和DoABCF2蛋白均包含2个保守的ABC结构域(分别为74~314、385~600和65~323、392~607)和多个基元;2个蛋白不含信号肽或跨膜域,预测均定位在叶绿体。2个基因与植物F家族ABC转运蛋白基因相似性高达80%以上,与玉米和水稻等单子叶植物F家族ABC转运蛋白基因亲缘关系较近。DoABCF1和DoABCF2基因转录本在石斛3个器官中差异表达且均在叶中高度表达,茎和根中相对表达量差异不显著;以茎为校正样本,前者在根中相对表达量为茎中的1.74倍,后者在叶中表达量为茎中的3.44倍。结论成功获得DoABCF1和DoABCF2基因全长cDNA,二者在铁皮石斛叶中的高表达特征暗示其可能在铁皮石斛生长发育过程中起一定作用。
[Abstract]:Objective to clone Dendrobium officinale ABC (ATP-binding cassette) transporter F gene and analyze it by bioinformatics. Methods ABC gene was isolated from the cDNA of Dendrobium candidum leaves by race, and the relative molecular weight, isoelectric point, domain, signal peptide, transmembrane domain and subcellular localization of the encoded protein were analyzed by bioinformatics. DNASTAR and MEGA6 were used for amino acid multi-sequence alignment and molecular evolution analysis, and real-time fluorescence quantitative PCR was used to detect the expression pattern of gene tissue. Results two F family ABC transporter genes, DoABCF1 and DoABCF2 (GenBank registration numbers KU160474 and KU160475), were isolated from Dendrobium candidum with a length of 2 104 BP and 2 193 BP, each encoding a peptide chain consisting of 600 and 659 amino acids. Relative molecular weight of 67 030 and 74 140, isoelectric points 6.20 and 5.71 of DoABCF1 and DoABCF2 proteins both contain two conserved ABC domains (74 / 314385 / 600 and 65 / 323392C / 607, respectively) and multiple motifs, and two proteins do not contain signal peptides or transmembrane domains. The similarity between the two genes and the ABC transporter genes of the F family of plants was more than 80%. The relationship between ABC transporter genes of F family in maize and rice was close to that of DoABCF1 and DoABCF2 gene transcripts in three organs of Dendrobium and were highly expressed in leaves, but there was no significant difference in relative expression between stem and root. The relative expression of the former in the root was 1.74 times that in the stem, and the latter in the leaf was 3.44 times of that in the stem. Conclusion the full length cDNAs of DoABCF1 and DoABCF2 genes were successfully obtained. Their high expression in the leaves of Dendrobium candidum suggests that they may play a role in the growth and development of Dendrobium candidum.
【作者单位】：陕西中医药大学药学院陕西省中药基础与新药研究重点实验室;陕西中医药大学附属医院第一临床医学院;中国医学科学院北京协和医学院药用植物研究所;
【基金】：国家自然科学基金项目(31101608) 陕西省自然科学基金项目(2017JM8030) 陕西省青年科技新星项目(2012KJXX-44)
【分类号】：Q943.2;S567.239

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